There are few published data concerning the induction and maintenance of immune tolerance based on concurrent clinical and mechanistic studies in rheumatoid arthritis (RA). It is of critical importance to understand the mechanisms of the induction of tolerance because the approach may be effective in maintaining disease control once the initial, symptomatic immune activation has been controlled by currently used therapies. We have recently concluded a NIH-sponsored placebo-controlled pilot phase II trial of mucosal tolerization to dnaJP1 in RA. dnaJP1 is a heat shock protein-derived peptide we previously identified as a contributor of T cell-mediated inflammation in RA. dnaJP1 treatment led to detectable clinical efficacy, which correlated with a significant reduction in the production of pro-inflammatory cytokine TNFa by T cells in response to dnaJP1 in vitro. Hypothesis-generating preliminary studies suggest that epitope-specific therapy affects T cell lineage commitment, resulting in a change from a pro-inflammatory (TH-1 and TH-17) to regulatory function. This is particularly evident in patients pre-treated with hydroxychloroquine (HCQ). These patients had a better clinical response to the dnaJP1 treatment. Pre-treatment with HCQ may induce a novel type of regulatory T cells (Treg), whose presence is necessary for the induction of tolerance. Therefore the hypotheses for this study are: i) epitope-specific therapy has a direct effect on the relative representation and function of T cell lineages within the gradient comprised from TH-1/TH-17 pro-inflammatory effector T cells (Teff) to Treg/ tolerogenic cells;ii) changes in the APC are induced by treatment with HCQ and are prodromic to the generation of a novel type of regulatory T cells, which is identified by the expression of PD-1;iii) The effects of therapy are initially peptide-specific and subsequently affect both innate and adaptive immunity. The objectives are: i) To demonstrate that therapy induces the emergence of various types of T cells with regulatory function that: a) act as suppressors for effector cells, thus favoring an immune deviation;b) can directly lyse antigen presenting cells (APC) which at the same time present dnaJP1 on their HLA and express the appropriate ligands (i.e. express PD-1 ligands to bind PD-1 Treg);ii) To characterize the nature and entity of the therapy-induced immune deviation in Teff from TH-1/TH-17 to a more tolerogenic phenotype;iii) To characterize the "adjuvant" role that HCQ has on the mechanism of tolerization, with a specific focus on HCQ-induced changes in APC phenotype and function. Specific Aim 1: To identify sub-populations of Treg based on co-expression of PD-1, characterize their function in relationship to immune tolerization to dnaJP1, and evaluate their suppressor/regulatory activity on Teff as well as on APC. Specific Aim 2: To characterize the nature and mechanisms of the treatment-induced immune deviation of Teff from pro- inflammatory TH-1/TH-17 to a tolerogenic status. Specific Aim 3: To study treatment-induced changes in APC function, with a specific focus on the "adjuvant" role that HCQ may have in facilitating the expression of a membrane phenotype capable of inducing and engaging T cell with a regulatory/suppressor potential. Our program is developing a novel approach that directly addresses the need of a tolerogen as a complement to currently used biologics. The research plan proposed here relies on a "bedside back to bench" reverse translational itinerary and is a fundamental part of the project, as it will dissect and define the mechanisms of tolerance from a unique collection of samples already obtained from a NIH-funded Phase II trial. These studies have the potential to provide valuable information regarding the immunology of human immune tolerance and will also help translate basic immunology concepts into novel tools for designing better trials and predicting treatment efficacy to optimize a patient's care regimen.